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protocol
Profiling DNA methylome landscapes of mammalian
cells with single-cell reduced-representation
bisulfite sequencing
Hongshan Guo1,5, Ping Zhu1,2,5, Fan Guo1, Xianlong Li1, Xinglong Wu1,2, Xiaoying Fan1, Lu Wen1,3,4 &
Fuchou Tang1,3,4


1Biodynamic Optical Imaging Center, College of Life Sciences, Peking University, Beijing, China. 2Peking-Tsinghua Center for Life Sciences, Peking University, Beijing,
China. 3Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, College of Life Sciences, Peking University, Beijing, China. 4Center for Molecular
and Translational Medicine, Peking University Health Science Center, Beijing, China. 5These authors contributed equally to this work. Correspondence should be
addressed to L.W. (wenlu.wl@gmail.com) or F.T. (tangfuchou@pku.edu.cn).

Published online 2 April 2015; doi:10.1038/nprot.2015.039

The heterogeneity of DNA methylation within a population of cells necessitates DNA methylome profiling at single-cell resolution.
Recently, we developed a single-cell reduced-representation bisulfite sequencing (scRRBS) technique in which we modified
the original RRBS method by integrating all the experimental steps before PCR amplification into a single-tube reaction. These
modifications enable scRRBS to provide digitized methylation information on ~1 million CpG sites within an individual diploid
mouse or human cell at single-base resolution. Compared with the single-cell bisulfite sequencing (scBS) technique, scRRBS covers
fewer CpG sites, but it provides better coverage for CpG islands (CGIs), which are likely to be the most informative elements forreserved. DNA methylation. The entire procedure takes ~3 weeks, and it requires strong molecular biology skills.
rights
All INTRODUCTION
DNA methylation of cytosine is a well-known epigenetic modi but it still requires three purifications after the end repair/dA
Inc. fication that is involved in gene expression regulation1. In mam tailing, the adapter ligation and the bisulfite conversion. In the
mals, DNA methylation is relatively stable in differentiated cells; scRRBS method, we integrated all the five steps into a single-
by contrast, global demethylation and remethylation occur tube reaction so that DNA purification does not occur until
during early embryonic development and primordial germ cell the completion of the bisulfite conversion17 (Fig. 1). To achieveAmerica,
development2–17. Whole-genome bisulfite sequencing provides this, the buffer system and the reaction volumes were modified
a comprehensive view of the DNA methylome, but it is very to preserve the different enzyme activities at each stage of theNature expensive owing to deep sequencing of the entire genome18–25. one-tube reaction.
An alternative and cost-efficient technique is RRBS26–28, in which When starting with a single mouse diploid cell, scRRBS covers,
2015 genomic DNA is first digested with a restriction endonuclease on average, 1 million CpG dinucleotides; this accounts for ~40%
© (usually MspI) and then size-selected to enrich for CpG-dense of the CpG sites that can be recovered by standard RRBS using
regions. RRBS provides comprehensive DNA methylation infor thousands of cells (on average, 2.5 million CpG dinucleotides).
mation about the genome. By sequencing ~10% of the mouse or Importantly, ~70% of CGIs in the mouse genome can be cap
human genome, RRBS can reproducibly cover a large proportion tured. The coverage increases with the number of starting cells,
of the informative CpG sites in the genome, including >70% of and it reaches a plateau at five cells that corresponds to 60% of
promoters, >80% of CGIs and a large number of CGI shores, the coverage achieved using standard RRBS, suggesting that the
enhancers, exons, 3′ untranslated regions (UTRs) and repetitive single-tube approach to some extent affects the coverage that can
elements27,29. However, conventional RRBS techniques require be achieved from the standard RRBS.
nanogram amounts of genomic DNA as starting material27,30,31,
and they are not applicable to single cells. Overview of the procedure
The scRRBS approach starts with cell picking and lysis in 5 µl
Development of the procedure of lysis buffer, which contains protease to release the genomic
We recently developed scRRBS for profiling DNA methylation DNA. The next three steps, including the MspI digestion, the
at the single-cell level10,17. In a typical mammalian cell, there end repair/dA tailing, and the adapter ligation, are accomplished
are only two copies of DNA molecules, and each comes from by adding the corresponding reaction components sequentially,
one of the two parents. If aiming to profile the DNA methy and the enzymes in the preceding reactions are inactivated by
lome of a single cell, it is crucial to avoid DNA loss as far as heating; Tango buffer is used for all the reactions. The ligation
possible, as all DNA fragments contain irreplaceable informa is performed by overnight incubation using the premethylated
tion. DNA loss is inevitable during purification steps before PCR sequencing adapters and highly concentrated T4 DNA ligase. The
amplification, and there are five such steps in the standard RRBS ligated DNA fragments are directly processed until bisulfite con
method: (i) genomic DNA purification; (ii) restriction enzyme version, and only after this step is the DNA purified in the pres
digestion; (iii) end repair and dA tailing (end repair/dA tailing); ence of carrier tRNA. Next, the DNA is PCR-amplified and the
(iv) adapter ligation; and (v) bisulfite conversion. A more recent fragments between 200 and 700 bp are gel-selected and purified
gel-free RRBS protocol reduces the number of purification steps, as the final library for sequencing.

nature protocols \| VOL.10 NO.5 \| 2015 \| 645

## Page 2

protocol

dynamics10. In addition, application of the technique to mouse
embryonic cells using as few as 20 cells faithfully captured the
CCGG CCGG Mspl digestion DNA methylation status of imprinted genomic regions at...
Picking a single cell methylation level of 50% (ref. 34).
(Steps 1–9)

End-repair/dA-tailing Limitations of single-cell methylation profiling techniques
adapter ligation
(Steps 13–19) The first limitation of the scRRBS technique stems from the design
of the RRBS method; that is, although it captures a large portion
of CGIs and promoters, it provides representative, but lower,
U U coverage of CpG-sparse regions such as enhancers27. It should
Single-tube reaction Bisulfite conversion be noted that the scBS technique is also biased toward CpG-rich U U (Steps 20–23)
genomic regions32, and there are currently no single-cell DNA
methylation profiling techniques that provide whole-genome
coverage. Second, both the scRRBS and the scBS techniques face
PCR amplification
(Steps 24–41) the issue of limited overlapping coverage between individual cells.
As mentioned above, the scBS approach suffers more from this
issue. Third, neither approach can discriminate between DNA
methylation (5mC) and hydroxymethylation (5hmC), and the
A detected methylation information is in fact the sum of 5mC
T High-throughput sequencing and 5hmC. Fourth, neither technique has been adapted to high-
G (Steps 42–44) throughput platforms such as the microfluidic chip. Fifth, the
C mapping efficiencies of both scRRBS and scBS are relatively lowreserved.
(~25% on average) when starting with a single cell. For scRRBS,
Figure 1 \| Flowchart of the experimental procedures of the scRRBS the mapping efficiency increases with the number of startingrights technique. Notably, we integrated cell lysis, MspI digestion, end repair/dA ...
tailing, adapter ligation and bisulfite treatment into a single-tube reaction
All standard RRBS17. to avoid unnecessary DNA loss.
Inc. Experimental design
Starting material. We have successfully applied this protocol to a
Comparison of scRRBS and single-cell bisulfite sequencing variety of mammalian cells, including human and mouse embry
Recently, Smallwood et al.32 reported an alternative scBS tech onic stem cells (mESCs), oocytes, sperm, early preimplantationAmerica, nique. The authors used the single-tube reaction strategy to ...
improve a previous protocol that was based on bisulfite conver ble to most mouse and human cell types, regardless of whether
sion followed by random priming33. When starting with a sinNature they are isolated from tissues or cell culture. However, it is rec
gle mouse diploid cell, scBS covers, on average, 3.7 million CpG ommended to select the healthiest-looking cells with good

protocol

Profiling DNA methylome landscapes of mammalian

cells with single-cell reduced-representation

bisulfite sequencing

Hongshan Guo1,5, Ping Zhu1,2,5, Fan Guo1, Xianlong Li1, Xinglong Wu1,2, Xiaoying Fan1, Lu Wen1,3,4 &

Fuchou Tang1,3,4

1Biodynamic Optical Imaging Center, College of Life Sciences, Peking University, Beijing, China. 2Peking-Tsinghua Center for Life Sciences, Peking University, Beijing,

China. 3Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, College of Life Sciences, Peking University, Beijing, China. 4Center for Molecular

and Translational Medicine, Peking University Health Science Center, Beijing, China. 5These authors contributed equally to this work. Correspondence should be

addressed to L.W. (wenlu.wl@gmail.com) or F.T. (tangfuchou@pku.edu.cn).

Published online 2 April 2015; doi:10.1038/nprot.2015.039

The heterogeneity of DNA methylation within a population of cells necessitates DNA methylome profiling at single-cell resolution.

Recently, we developed a single-cell reduced-representation bisulfite sequencing (scRRBS) technique in which we modified

the original RRBS method by integrating all the experimental steps before PCR amplification into a single-tube reaction. These

modifications enable scRRBS to provide digitized methylation information on ~1 million CpG sites within an individual diploid

mouse or human cell at single-base resolution. Compared with the single-cell bisulfite sequencing (scBS) technique, scRRBS covers

fewer CpG sites, but it provides better coverage for CpG islands (CGIs), which are likely to be the most informative elements forreserved. DNA methylation. The entire procedure takes ~3 weeks, and it requires strong molecular biology skills.

rights

All INTRODUCTION

DNA methylation of cytosine is a well-known epigenetic modi but it still requires three purifications after the end repair/dA

Inc. fication that is involved in gene expression regulation1. In mam tailing, the adapter ligation and the bisulfite conversion. In the

mals, DNA methylation is relatively stable in differentiated cells; scRRBS method, we integrated all the five steps into a single-

by contrast, global demethylation and remethylation occur tube reaction so that DNA purification does not occur until

during early embryonic development and primordial germ cell the completion of the bisulfite conversion17 (Fig. 1). To achieveAmerica,

development2–17. Whole-genome bisulfite sequencing provides this, the buffer system and the reaction volumes were modified

a comprehensive view of the DNA methylome, but it is very to preserve the different enzyme activities at each stage of theNature expensive owing to deep sequencing of the entire genome18–25. one-tube reaction.

An alternative and cost-efficient technique is RRBS26–28, in which When starting with a single mouse diploid cell, scRRBS covers,

2015 genomic DNA is first digested with a restriction endonuclease on average, 1 million CpG dinucleotides; this accounts for ~40%

regions. RRBS provides comprehensive DNA methylation infor thousands of cells (on average, 2.5 million CpG dinucleotides).

mation about the genome. By sequencing ~10% of the mouse or Importantly, ~70% of CGIs in the mouse genome can be cap

human genome, RRBS can reproducibly cover a large proportion tured. The coverage increases with the number of starting cells,

of the informative CpG sites in the genome, including >70% of and it reaches a plateau at five cells that corresponds to 60% of

promoters, >80% of CGIs and a large number of CGI shores, the coverage achieved using standard RRBS, suggesting that the

enhancers, exons, 3′ untranslated regions (UTRs) and repetitive single-tube approach to some extent affects the coverage that can

elements27,29. However, conventional RRBS techniques require be achieved from the standard RRBS.

nanogram amounts of genomic DNA as starting material27,30,31,

and they are not applicable to single cells. Overview of the procedure

The scRRBS approach starts with cell picking and lysis in 5 µl

Development of the procedure of lysis buffer, which contains protease to release the genomic

We recently developed scRRBS for profiling DNA methylation DNA. The next three steps, including the MspI digestion, the

at the single-cell level10,17. In a typical mammalian cell, there end repair/dA tailing, and the adapter ligation, are accomplished

are only two copies of DNA molecules, and each comes from by adding the corresponding reaction components sequentially,

one of the two parents. If aiming to profile the DNA methy and the enzymes in the preceding reactions are inactivated by

lome of a single cell, it is crucial to avoid DNA loss as far as heating; Tango buffer is used for all the reactions. The ligation

possible, as all DNA fragments contain irreplaceable informa is performed by overnight incubation using the premethylated

tion. DNA loss is inevitable during purification steps before PCR sequencing adapters and highly concentrated T4 DNA ligase. The

amplification, and there are five such steps in the standard RRBS ligated DNA fragments are directly processed until bisulfite con

method: (i) genomic DNA purification; (ii) restriction enzyme version, and only after this step is the DNA purified in the pres

digestion; (iii) end repair and dA tailing (end repair/dA tailing); ence of carrier tRNA. Next, the DNA is PCR-amplified and the

(iv) adapter ligation; and (v) bisulfite conversion. A more recent fragments between 200 and 700 bp are gel-selected and purified

gel-free RRBS protocol reduces the number of purification steps, as the final library for sequencing.

nature protocols | VOL.10 NO.5 | 2015 | 645

## Page 2

protocol

dynamics10. In addition, application of the technique to mouse

embryonic cells using as few as 20 cells faithfully captured the

CCGG CCGG Mspl digestion DNA methylation status of imprinted genomic regions at...

Picking a single cell methylation level of 50% (ref. 34).

(Steps 1–9)

End-repair/dA-tailing Limitations of single-cell methylation profiling techniques

adapter ligation

(Steps 13–19) The first limitation of the scRRBS technique stems from the design

of the RRBS method; that is, although it captures a large portion

of CGIs and promoters, it provides representative, but lower,

U U coverage of CpG-sparse regions such as enhancers27. It should

Single-tube reaction Bisulfite conversion be noted that the scBS technique is also biased toward CpG-rich U U (Steps 20–23)

genomic regions32, and there are currently no single-cell DNA

methylation profiling techniques that provide whole-genome

coverage. Second, both the scRRBS and the scBS techniques face

PCR amplification

(Steps 24–41) the issue of limited overlapping coverage between individual cells.

As mentioned above, the scBS approach suffers more from this

issue. Third, neither approach can discriminate between DNA

methylation (5mC) and hydroxymethylation (5hmC), and the

A detected methylation information is in fact the sum of 5mC

T High-throughput sequencing and 5hmC. Fourth, neither technique has been adapted to high-

G (Steps 42–44) throughput platforms such as the microfluidic chip. Fifth, the

C mapping efficiencies of both scRRBS and scBS are relatively lowreserved.

(~25% on average) when starting with a single cell. For scRRBS,

Figure 1 | Flowchart of the experimental procedures of the scRRBS the mapping efficiency increases with the number of startingrights technique. Notably, we integrated cell lysis, MspI digestion, end repair/dA ...

tailing, adapter ligation and bisulfite treatment into a single-tube reaction

All standard RRBS17. to avoid unnecessary DNA loss.

Inc. Experimental design

Starting material. We have successfully applied this protocol to a

Comparison of scRRBS and single-cell bisulfite sequencing variety of mammalian cells, including human and mouse embry

Recently, Smallwood et al.32 reported an alternative scBS tech onic stem cells (mESCs), oocytes, sperm, early preimplantationAmerica, nique. The authors used the single-tube reaction strategy to ...

improve a previous protocol that was based on bisulfite conver ble to most mouse and human cell types, regardless of whether

sion followed by random priming33. When starting with a sinNature they are isolated from tissues or cell culture. However, it is rec

gle mouse diploid cell, scBS covers, on average, 3.7 million CpG ommended to select the healthiest-looking cells with good